Feed Composition for Improving Intraintestinal Environment

ABSTRACT

A feed composition for improving intestinal environment by promoting proliferation of intestinal bifidobacteria is described herein. The feed composition for improving intestinal environment can contain a combination of (1) an enteric composition containing an amino acid or a salt thereof, and (2) an indigestible polysaccharide or a composition containing an indigestible polysaccharide.

This application is a continuation of, and claims priority under 35 U.S.C. § 120 to, International Application No. PCT/JP2018/013054, filed Mar. 28, 2018, and claims priority therethrough under 35 U.S.C. § 119 to Japanese Patent Application No. 2017-063145, filed Mar. 28, 2017, the entireties of which are incorporated by reference herein. Also, the Sequence Listing filed electronically herewith is hereby incorporated by reference (File name: 2019-09-23T US-602 Seq_List; File size: 1 KB; Date recorded: Sep. 23, 2019).

BACKGROUND Technical Field

The present invention relates to a feed composition for improving the intestinal environment by promoting proliferation of intestinal bifidobacteria.

Review of Related Art

It is important for prevention of various diseases and for the promotion and maintenance of the health of mammals, birds, fish, and crustaceans such as livestock, poultry, and laboratory animals, to promote proliferation of beneficial bacteria such as intestinal bifidobacteria and the like and improve the intestinal environment by normalization of the intestinal bacterial flora.

National Publication of International Patent Application No. 2009-517032 discloses a sweetener composition containing prebiotics, and describes that the prebiotics include mucopolysaccharides, oligosaccharides, polysaccharides, amino acids, vitamins, nutrient precursors, proteins, and combinations of these. However, the document does not demonstrate that the above-mentioned prebiotics cause proliferation of bifidobacteria in the gastrointestinal tract via experimental data or by suggestion.

“Bifidobacteria Research” Tomotari MITSUOKA (Center for Academic Publications Japan, 1998, P70) reports on the amino acid requirements of bifidobacteria. However, this report shows experimental results of a pure culture strain using a medium richer in nutrients than in the intestine, and no studies have been reported to date of intestinal bacterial flora in a complex system such as the lower gastrointestinal tract or of being oligotrophic.

It is known that polysaccharides such as oligosaccharides and water-soluble dietary fibers such as inulin are difficult to digest and be absorbed in the stomach and small intestine, and hence reach the large intestine to become bait for beneficial bacteria such as bifidobacteria and the like (e.g., GIBSON et al., GASTROENTEROLOGY, Vol. 108, No. 4, pages 975-982, 1995; Kolida et al., Br J Nutr. 2002 May; 87 Suppl 2: S193-197).

SUMMARY

The present invention provides a feed composition useful for improving the intestinal environment by promoting proliferation of intestinal bifidobacteria.

It has been found from the results of the Experimental Examples 1-1, 1-2 and 2 (culture test using human feces suspension) and further from the results of Experimental Example 3 (administration test on pigs) that a remarkably superior promoting effect on the proliferation of bifidobacteria is obtained by supplying a combination of oligosaccharides (indigestible polysaccharides) and amino acids as an active ingredient as compared to when each is used alone.

In addition, amino acids are generally absorbed in the jejunum and upper ileum, and therefore, they do not easily travel to the lower gastrointestinal tract where the bifidobacteria live. However, it has been found that oligosaccharides, such as indigestible polysaccharide, and amino acids can be simultaneously present in the lower gastrointestinal tract by forming an enteric composition of the amino acid.

It is an aspect of the present invention to provide a feed composition for improving an intestinal environment comprising: (1) an enteric composition comprising an amino acid or a salt thereof, and (2) an indigestible polysaccharide or a composition comprising an indigestible polysaccharide.

It is a further aspect of the present invention to provide the feed composition as described above, wherein the indigestible polysaccharide is an oligosaccharide.

It is a further aspect of the present invention to provide the feed composition as described above, wherein the amino acid is selected from the group consisting of alanine, glutamine, glutamic acid, isoleucine, histidine, lysine, proline, valine, and combinations thereof.

It is a further aspect of the present invention to provide the feed composition as described above, wherein the amino acid is selected from the group consisting of alanine, glutamine, glutamic acid, isoleucine, lysine, valine, and combinations thereof.

It is a further aspect of the present invention to provide the feed composition as described above, wherein the amino acid is selected from the group consisting of glutamic acid, lysine, and a combination thereof.

It is a further aspect of the present invention to provide the feed composition as described above, wherein the form of the enteric composition is selected from the group consisting of a granule, a granulated substance, a tablet, a hard capsule, and a soft capsule.

It is a further aspect of the present invention to provide the feed composition as described above, wherein the form of the composition comprising an oligosaccharide is a drink.

It is as aspect of the present invention to provide a commercial package comprising the feed composition as described above, and written matter describing and explaining a method for improving the intestinal environment using said feed composition.

It is a further aspect of the present invention to provide a feed composition with an indication that it can be used for improving intestinal environment, comprising the feed composition as described above.

It is a further aspect of the present invention to provide a feed composition for enhancing an intestinal environment improving effect comprising the feed composition as described above.

It is a further aspect of the present invention to provide a feed composition for enhancing an intestinal environment improving effect of an indigestible polysaccharide or a composition comprising an indigestible polysaccharide, said feed composition being an enteric composition comprising an amino acid or a salt thereof.

It is a further aspect of the present invention to provide the feed composition as described above, said feed composition being used for improving the intestinal environment.

It is a further aspect of the present invention to provide a method for improving the intestinal environment in non-human mammals, birds, fish, or crustaceans comprising ingestion by the non-human mammals, birds, fish, or crustaceans of an effective amount of a the feed composition as described above.

It is a further aspect of the present invention to provide a method of producing a feed composition for improving the intestinal environment comprising combining (1) an enteric composition comprising an amino acid or a salt thereof, and (2) an indigestible polysaccharide or a composition comprising an indigestible polysaccharide.

It is a further aspect of the present invention to provide a feed composition for improving intestinal environment comprising: (1) an enteric composition comprising an amino acid, and (2) an oligosaccharide or a composition comprising an oligosaccharide.

It is a further aspect of the present invention to provide the feed composition as described above, wherein the amino acid is selected from the group consisting of alanine, glutamine, glutamic acid, isoleucine, lysine, valine, and combinations thereof.

It is a further aspect of the present invention to provide the feed composition as described above, wherein the amino acid is selected from the group consisting of glutamic acid, lysine, and a combination thereof.

It is a further aspect of the present invention to provide the feed composition as described above, wherein the form of the enteric composition is selected from the group consisting of a granule, a tablet, a hard capsule, and a soft capsule.

It is a further aspect of the present invention to provide the feed composition as described above, wherein the composition comprising an oligosaccharide is a drink.

It is a further aspect of the present invention to provide a commercial package comprising a feed composition comprising: (1) an enteric composition comprising an amino acid, and (2) an oligosaccharide or a composition comprising an oligosaccharide, and written matter describing and explaining a method for improving the intestinal environment using the feed composition.

It is a further aspect of the present invention to provide a feed composition with an indication that it can be used for improving an intestinal environment, comprising: (1) an enteric composition comprising an amino acid, and (2) an oligosaccharide or a composition comprising an oligosaccharide.

It is a further aspect of the present invention to provide a feed composition for enhancing an intestinal environment improving effect comprising: (1) an enteric composition comprising an amino acid, and (2) an oligosaccharide or a composition comprising an oligosaccharide.

It is a further aspect of the present invention to provide a feed composition for enhancing an intestinal environment improving effect of a composition comprising an oligosaccharide or a composition comprising an oligosaccharide, said feed composition being an enteric composition comprising an amino acid.

As described herein, a feed composition having a remarkably superior promoting effect on the proliferation of bifidobacteria as compared to when each is used singly and a function to improve the intestinal environment can be provided by supplying a combination of an indigestible polysaccharide, such as oligosaccharide or water-soluble dietary fiber, and an amino acid or a salt thereof.

As described herein, by supplying a combination of an amino acid or a salt thereof as an enteric composition and an indigestible polysaccharide, such as an oligosaccharide, or water-soluble dietary fiber), the indigestible polysaccharide and the amino acid or a salt thereof can be simultaneously present in the lower gastrointestinal tract where bifidobacteria live, and proliferation of intestinal bifidobacteria can be promoted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-1 shows the results of Experimental Example 1-1.

FIG. 1-2 shows the results of Experimental Example 1-2.

FIG. 2 shows the results of Experimental Example 2.

FIG. 3 shows the results of relative amounts of cecal bifidobacteria in Experimental Example 3.

FIG. 4 shows the results of cecal lysine concentration in Experimental Example 3.

DETAILED DESCRIPTION

The feed composition for improving intestinal environment as described herein is composed of a combination of (1) an enteric composition containing an amino acid or a salt thereof, and (2) an indigestible polysaccharide or a composition containing an indigestible polysaccharide.

The feed composition is a combined-use feed composition of (1) and (2) in which (1) and (2) are used in combination.

In the feed composition as described herein, (1) and (2) may be simultaneously formulated and contained in the same preparation (feed composition), or (1) and (2) may be formulated separately and ingested simultaneously or at different times by the same route or different routes. That is, the feed composition as described herein includes a feed composition containing (1) and (2) in one preparation, and a feed composition combining (1) and (2) formulated separately.

The feed composition as described herein is a concept that broadly encompasses compositions that can be ingested orally, excluding pharmaceutical products, and includes not only so-called livestock feeds such as blended feed, mixed feed, premix, and the like, but also feed for pets, health supplements (health supplement feed), supplements, drinks, and the like.

The phrase “improvement of intestinal environment” can refer to increasing the relative bifidobacteria present in the lower gastrointestinal tract. To relatively increase can mean to promote the predominance of bifidobacteria in the intestinal bacterial flora. The predominance of bifidobacteria in the intestinal bacterial flora can be confirmed by measuring the amount of bifidobacteria in feces by quantitative PCR or the like.

The “lower gastrointestinal tract” can mean the ileum, cecum, colon, and rectum.

(1) Enteric Composition Containing Amino Acid or a Salt Thereof

Examples of the amino acid include alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and valine; and alanine, glutamine, glutamic acid, isoleucine, histidine, lysine, proline, and valine are particular examples. These amino acids can be in an L form. One or more of these amino acids can be used in combination.

As the amino acid, L-alanine, L-glutamine, L-glutamic acid, L-isoleucine, L-histidine, L-lysine, L-proline, L-valine or a combination of these, such as a combination of L-glutamic acid and L-lysine is preferable, and L-glutamic acid, L-lysine, or a combination of L-glutamic acid and L-lysine is more another particular example.

The amino acid may be in the form of a salt. Examples include salts acceptable as a feed. Examples include alkali metal salts such as sodium salt, potassium salt, and the like; alkaline earth metal salts such as calcium salt, magnesium salt, barium salt, and the like; aluminum salt; salts with organic bases such as ethylenediamine, propylenediamine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, triethanolamine, and the like; salts with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, and the like; and salts with organic acids such as formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like can be mentioned, and sodium salt, potassium salt or hydrochloride is a particular example.

The enteric composition is a composition that does not disintegrate in the stomach or the upper part of the small intestine where the pH is low, but disintegrates in the lower part of the small intestine or large intestine where the pH is high and the amino acid or a salt thereof can be effective.

The enteric composition can disintegrate at pH 4 or more, pH 4-pH 9, or pH 5-pH 9.

The disintegration property can be measured, for example, according to the rules for enteric preparations in the disintegration test method, the Japanese Pharmacopoeia 17th edition.

Examples of the form of the enteric composition include granules, including fine granules, a granulated substance, tablet, hard capsule, and soft capsule.

The enteric composition can be produced by a method known in the field of feed preparation, food preparation, or pharmaceutical preparation.

For example, methods of production can include mixing, granulating and/or tableting an amino acid or a salt thereof together with a carrier, such as an excipient, binder, disintegrant, lubricant, protector, to give a granule or tablet, then applying an enteric coating to the granule or tablet to give an enteric granule or enteric tablet; filling granules or tablets containing an amino acid or a salt thereof in a hard capsule already having an enteric coating to give an enteric capsule; and encapsulating a suspension of an amino acid or a salt thereof in a carrier, such as an oil component, in a soft capsule composed of an enteric substrate to give an enteric capsule. In addition, the above-mentioned enteric granules may be encapsulated in a capsule, such as a cellulose capsule to give a capsule. Also, a molten mixture of oil and fat (protector) and amino acid or a salt thereof may be solidified by cooling to give an enteric granulated substance.

In addition, commercially available products, such as the lysine formulation for dairy cows “AjiPro-L” manufactured by Ajinomoto Co., Inc., can also be used.

Examples of the enteric coating base include cellulose polymers such as hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, carboxymethylethylcellulose, cellulose acetate phthalate, and the like; acrylic acid polymers such as methacrylic acid copolymer L, methacrylic acid copolymer LD, methacrylic acid copolymer S, and the like; and naturally occurring substances such as shellac and the like. A coating additive such as a plasticizer may also be used during coating.

The carrier, coating base and coating additive are used in amounts conventionally employed in the technical field of preparation formulation.

(2) Indigestible Polysaccharide or Composition Containing Indigestible Polysaccharide

The indigestible polysaccharide is a polysaccharide that is not easily digested or absorbed in the stomach or small intestine, and therefore, that reaches the large intestine to be utilized by bifidobacteria.

The degree of polymerization of the indigestible polysaccharides is not less than 2 or not less than 3. The upper limit of the degree of polymerization of indigestible polysaccharides is not particularly limited and can be, for example, not more than 100, not more than 60, not more than 10, not more than 6, or not more than 5.

The degree of polymerization of the indigestible polysaccharides can be 2-100, 2-60, 2-10, or 2-6.

Examples of the indigestible polysaccharides include oligosaccharides, water-soluble dietary fiber, and the like.

Indigestible polysaccharides, such as oligosaccharides or water-soluble dietary fiber, can be used as they are or they can be in a composition containing indigestible polysaccharides.

The oligosaccharide can have 2 to 10 monosaccharides bonded thereto.

Examples of the oligosaccharide include coffee bean manno-oligosaccharide, lactosucrose oligosaccharide, galacto-oligosaccharide, fructo-oligosaccharide, soybean oligosaccharide, xylo-oligosaccharide, and isomalto-oligosaccharide; and fructo-oligosaccharide or galacto-oligosaccharide is a particular example. One or more kinds of oligosaccharides can be used in combination.

The oligosaccharide can be produced by a known method, and commercially available products can also be used.

Examples of the fructo-oligosaccharide include fructo-oligosaccharides with polymerization degree of 3-5 in which 1 to 3 fructoses are bonded to sucrose. Specifically, for example, FOS: Wako Pure Chemical Industries, Ltd., #064-02385 is a particular example.

Examples of the galacto-oligosaccharide include galacto-oligosaccharide with polymerization degree of 2-6 in which galactose is bonded to lactose. Specifically, for example, GOS: Wako Pure Chemical Industries, Ltd., #076-05945 is a particular example.

Water-soluble dietary fiber is a dietary fiber that is not easily digested or absorbed in the stomach or small intestine, and can reach the large intestine to be degraded by intestinal bacteria and utilized by bifidobacteria.

Examples of the water-soluble dietary fiber include inulin.

The water-soluble dietary fiber such as inulin can be produced by a known method, and commercially available products can also be used.

Inulin generally has a structure in which 2-60 molecules of fructose are polymerized by a β(2-1) bond and a glucose molecule is bonded to the terminal.

Inulin is degraded by intestinal bacteria in the large intestine (partial hydrolysis) to be fructo-oligosaccharide. Thus, an effect similar to the effect of fructo-oligosaccharide was confirmed in the Experimental Examples 1-1, 1-2, and is expected to be present in the gastrointestinal tract.

The form of the composition containing indigestible polysaccharides, such as an oligosaccharide or water-soluble dietary fiber is not particularly limited and may be, for example, a powder, granule (including fine granules), tablet, hard capsule, soft capsule, liquid (e.g., solution, suspension, milky lotion), drink, jelly, pudding, yogurt, candy, chewing gum, or the like. These can be produced by a known method. For example, indigestible polysaccharides, such as an oligosaccharide or water-soluble dietary fiber are mixed with carriers, such as an excipient, binder, disintegrant, lubricant, solvent, to produce a powder, granule, tablet, capsule, liquid, and the like by a method known in the field of feed preparation, food preparation, or pharmaceutical preparation. In addition, they can also be produced by adding and mixing indigestible polysaccharides, such as an oligosaccharide or water-soluble dietary fiber to and with feed and drink, such as water or a soft drink. Commercially available products, such as fructo-oligosaccharide “Meiorigo P granule” manufactured by Meiji Food Materia Co., Ltd. can also be used.

As described above, the feed composition as described herein includes a feed composition containing the above-mentioned (1) and (2) in one preparation, and a feed composition combining the above-mentioned (1) and (2) formulated separately.

As the feed composition combining (1) and (2) formulated separately, for example, a feed composition is described combining (1) in the form of an enteric granule, enteric granulated substance, enteric tablet, enteric hard capsule, or enteric soft capsule containing an amino acid or a salt thereof, and (2) in the form of a drink containing an indigestible polysaccharide, such as an oligosaccharide or water-soluble dietary fiber.

(1) and (2) may be ingested simultaneously or at different times by the same route or different routes. Specifically, for example, an enteric capsule, such as a hard capsule or soft capsule containing an amino acid or a salt thereof, enteric granule, enteric granulated substance, and the like may be ingested together with a drink containing an indigestible polysaccharide, such as an oligosaccharide or water-soluble dietary fiber.

The ingestion amount of an amino acid or a salt thereof in the feed composition for improving intestinal environment varies depending on the kind of animal and the like. For example, it can be 0.03-6 g/kg body weight based on amino acid per day.

The ingestion amount of an indigestible polysaccharide such as an oligosaccharide or water-soluble dietary fiber in the feed composition for improving the intestinal environment varies depending on the kind of animal and the like. For example, it can be 0.06-40 g/kg body weight per day.

For example, in the case of a pig weighing about 35 kg, the ingestion amount of an amino acid or a salt thereof can be 1-50 g, or 5-15 g, per day as an amino acid. The ingestion amount of indigestible polysaccharides such as an oligosaccharide or water-soluble dietary fiber can be 2-200 g, or 20-60 g, per day.

The feed composition can be safely ingested by animals other than human, for example, non-human mammals such as cows, pigs, horses, sheep, goats, monkeys, mice, rats, hamsters, rabbits, cats, and dogs; birds such as chickens, ducks, turkeys, quails, and ostriches; fish especially cultivated fish such as salmon trout, carp, red sea bream, eel, young yellowtail, great amberjack, flounder, tuna, tilapia, and catfish; crustaceans especially cultivated crustaceans such as prawns, vaname prawns, black tigers; and other non-human mammals such as domestic animals, poultry, laboratory animals, pets, etc. The form of ingestion by animals other than human may be as an addition to a feed.

The subject to which the feed composition is applied can be a single stomach animal.

The feed composition can be supplied singly to non-human mammals, birds, fish, or crustaceans. However, it can also be added to and mixed with general feeds, and supplied to non-human mammals, birds, fish, or shellfish.

Examples of the general feeds include brans such as wheat bran, rice bran, barley bran, millet bran, and the like; food processing by-products such as soybean-curd residue, starch pulp, copra meal, sake cake, soy sauce cake, brewer's grains, sweet potato distiller's residue, juice pulp of fruits and vegetables, and the like; cereals such as corn, rice, wheat, barley, oat, rye, sorghum, soybean, toasted soybean flour, and the like, and their processed products; oil seed meals such as soybean meal, rapeseed meal, cotton seed meal, linseed meal, sesame meal, sunflower meal, and the like; animal origin feeds such as fish meal, casein, dried skim milk, skim milk, whey, dried whey, meat, and bone meal, meat meal, feather meal, blood meal, meat-and-bone meal, plasma protein, and the like; leaf meals such as alfalfa meal and the like; saccharides such as sugar and the like and other sweeteners; sodium chloride; fats and oils; soybean protein; and grass.

The feed composition may contain an excipient such as cellulose derivatives such as carboxymethyl cellulose, a bulking agent such as dextrin or starch, a nutritional reinforcing agent such as vitamins or minerals, a feed additive such as enzyme agents or viable bacteria agents, and the like as long as the effects of the feed composition are not impaired.

Further embodiments include a commercial package containing a feed composition containing a combination of (1) an enteric composition containing an amino acid or a salt thereof, and (2) an indigestible polysaccharide or a composition containing an indigestible polysaccharide, and written matter describing and explaining uses of the feed composition for improving the intestinal environment; a feed composition with an indication that it can be useful for improving the intestinal environment, which contains a combination of (1) an enteric composition containing an amino acid or a salt thereof, and (2) an indigestible polysaccharide or a composition containing an indigestible polysaccharide; a feed composition for enhancing an intestinal environment improving effect containing a combination of (1) an enteric composition containing an amino acid or a salt thereof, and (2) an indigestible polysaccharide or a composition containing an indigestible polysaccharide; and a feed composition for enhancing an intestinal environment improving effect of an indigestible polysaccharide or a composition containing an indigestible polysaccharide, which feed composition is an enteric composition containing an amino acid or a salt thereof. The “enteric composition containing an amino acid or a salt thereof”, “indigestible polysaccharide or a composition containing an indigestible polysaccharide”, “intestinal environment improvement”, “ingestion amount of an amino acid or a salt thereof” and “ingestion amount of an indigestible polysaccharides” are the same as exemplifications and definitions indicated for the above-mentioned feed composition for improving intestinal environment.

EXAMPLES

The present invention is explained in more detail in the following by referring to the following non-limiting Experimental Examples, Examples, Comparative Examples, and Reference Examples.

Abbreviations mean the following:

-   Ala: L-alanine -   Arg: L-arginine -   Asn: L-asparagine -   Cys: L-cysteine -   Gln: L-glutamine -   Glu: L-glutamic acid -   Gly: glycine -   His: L-histidine -   Ile: L-isoleucine -   Leu: L-leucine -   Lys: L-lysine -   Met: L-methionine -   Phe: L-phenylalanine -   Pro: L-proline -   Ser: L-serine -   Thr: L-threonine -   Val: L-valine -   FOS: fructo-oligosaccharide -   GOS: galacto-oligosaccharide -   PBS: phosphate buffered saline

Experimental Example 1-1: Addition Test of Amino Acid and Fructo-Oligosaccharide to Human Feces Suspension

The PBS solutions for the Examples and Comparative Examples shown in Table 1-1 were prepared and used as media for a feces culture. PBS alone was used as a control. Fructo-oligosaccharide (FOS) manufactured by Wako Pure Chemical Industries, Ltd. (#064-02385) was used.

Thereafter, 10 ml of each solution was dispensed into glass vials and the glass vials were sterilized by autoclave, made anaerobic, and sealed with a butyl rubber stopper. The feces suspension was prepared by collecting about 10 g of feces from 3 healthy individuals (one man and two women) in their 20s to 40 s and suspending same in 80 ml of anaerobic PBS. The feces suspension was added to each vial by 0.5 ml, placed in an incubator at 37° C., and anaerobically cultured for 4 days with tumble blending once per day. After completion of the culture, an intestinal bacterial pellet was obtained from the entire amount of the culture medium by centrifugation (8,000×g, 5 min). DNA was extracted from this pellet using ISOFECAL for BeadsBeat (manufactured by NIPPON GENE CO., LTD.), and quantitative PCR was performed using GeneAce SYBR qPCR Mix α (manufactured by NIPPON GENE CO., LTD.). Quantitative PCR was performed under the conditions of 45 cycles of 95° C. for 30 sec and 60° C. for 60 sec after 95° C. for 10 min. Primers for quantitative PCR used for detection bifidobacteria are as follows:

g-Bifid-F: (SEQ ID NO: 1) 5′-CTCCTGGAAACGGGTGG-3′ g-Bifid-R: (SEQ ID NO: 2) 5′-GGTGTTCTTCCCGATATCTACA-3′

TABLE 1-1 medium for feces culture Example 1-1 0.1% Ala and 0.5% FOS-containing PBS solution Example 1-2 0.5% Ala and 0.5% FOS-containing PBS solution Example 1-3 1.0% Ala and 0.5% FOS-containing PBS solution Example 2-1 0.1% Arg and 0.5% FOS-containing PBS solution Example 2-2 0.5% Arg and 0.5% FOS-containing PBS solution Example 2-3 1.0% Arg and 0.5% FOS-containing PBS solution Example 3-1 0.1% Gln and 0.5% FOS-containing PBS solution Example 3-2 0.5% Gln and 0.5% FOS-containing PBS solution Example 3-3 1.0% Gln and 0.5% FOS-containing PBS solution Example 4-1 0.1% Glu and 0.5% FOS-containing PBS solution Example 4-2 0.5% Glu and 0.5% FOS-containing PBS solution Example 4-3 1.0% Glu and 0.5% FOS-containing PBS solution Example 5-1 0.1% Gly and 0.5% FOS-containing PBS solution Example 5-2 0.5% Gly and 0.5% FOS-containing PBS solution Example 5-3 1.0% Gly and 0.5% FOS-containing PBS solution Example 6-1 0.1% Ile and 0.5% FOS-containing PBS solution Example 6-2 0.5% Ile and 0.5% FOS-containing PBS solution Example 6-3 1.0% Ile and 0.5% FOS-containing PBS solution Example 7-1 0.1% Lys and 0.5% FOS-containing PBS solution Example 7-2 0.5% Lys and 0.5% FOS-containing PBS solution Example 7-3 1.0% Lys and 0.5% FOS-containing PBS solution Example 8-1 0.1% Ser and 0.5% FOS-containing PBS solution Example 8-2 0.5% Ser and 0.5% FOS-containing PBS solution Example 8-3 1.0% Ser and 0.5% FOS-containing PBS solution Example 9-1 0.1% Val and 0.5% FOS-containing PBS solution Example 9-2 0.5% Val and 0.5% FOS-containing PBS solution Example 9-3 1.0% Val and 0.5% FOS-containing PBS solution Example 10-1 0.1% Glu, 0.1% Lys and 0.5% FOS-containing PBS solution Example 10-2 0.5% Glu, 0.5% Lys and 0.5% FOS-containing PBS solution Example 10-3 1.0% Glu, 1.0% Lys and 0.5% FOS-containing PBS solution Comparative 1.0% Ala-containing PBS solution Example 1 Comparative 1.0% Arg-containing PBS solution Example 2 Comparative 1.0% Gln-containing PBS solution Example 3 Comparative 1.0% Glu-containing PBS solution Example 4 Comparative 1.0% Gly-containing PBS solution Example 5 Comparative 1.0% Ile-containing PBS solution Example 6 Comparative 1.0% Lys-containing PBS solution Example 7 Comparative 1.0% Ser-containing PBS solution Example 8 Comparative 1.0% Val-containing PBS solution Example 9 Comparative 1.0% Glu and 1.0% Lys-containing Example 10 PBS solution Comparative 0.5% FOS-containing PBS solution Example 11 Reference PBS alone (control) Example 1

Using the results obtained by quantitative PCR, the relative amount when the control is 1 was calculated, and the results were normalized with z-score.

The results are shown in FIG. 1-1. The data shows a mean of z-score±standard error (n=3).

As a result, proliferation of bifidobacteria was not observed when any amino acid was added alone. However, when Ala, Gln, Glu, Ile, Lys, and Val were respectively added together with 0.5% FOS, greater proliferation of bifidobacteria than that with FOS alone was observed. In addition, when Glu and Lys were mixed and added together with 0.5% FOS, more prominent proliferation of bifidobacteria than that with FOS alone was similarly observed.

Thus, a synergistic effect exceeding the additive effect was observed in the proliferation of bifidobacteria by combining an amino acid and oligosaccharide.

Experimental Example 1-2: Addition Test of Amino Acid and Fructo-Oligosaccharide to Human Feces Suspension

The PBS solutions for the Examples and Comparative Examples shown in Table 1-2 were prepared and used as media for a feces culture. PBS alone was used as a control. Feces culture and detection of bifidobacteria were performed according to methods similar to those in Experimental Example 1-1.

TABLE 1-2 medium for feces culture Example 11-1 0.1% Asn and 0.5% FOS-containing PBS solution Example 11-2 0.5% Asn and 0.5% FOS-containing PBS solution Example 11-3 1.0% Asn and 0.5% FOS-containing PBS solution Example 12-1 0.1% Cys and 0.5% FOS-containing PBS solution Example 12-2 0.5% Cys and 0.5% FOS-containing PBS solution Example 12-3 1.0% Cys and 0.5% FOS-containing PBS solution Example 13-1 0.1% His and 0.5% FOS-containing PBS solution Example 13-2 0.5% His and 0.5% FOS-containing PBS solution Example 13-3 1.0% His and 0.5% FOS-containing PBS solution Example 14-1 0.1% Leu and 0.5% FOS-containing PBS solution Example 14-2 0.5% Leu and 0.5% FOS-containing PBS solution Example 14-3 1.0% Leu and 0.5% FOS-containing PBS solution Example 15-1 0.1% Met and 0.5% FOS-containing PBS solution Example 15-2 0.5% Met and 0.5% FOS-containing PBS solution Example 15-3 1.0% Met and 0.5% FOS-containing PBS solution Example 16-1 0.1% Phe and 0.5% FOS-containing PBS solution Example 16-2 0.5% Phe and 0.5% FOS-containing PBS solution Example 16-3 1.0% Phe and 0.5% FOS-containing PBS solution Example 17-1 0.1% Pro and 0.5% FOS-containing PBS solution Example 17-2 0.5% Pro and 0.5% FOS-containing PBS solution Example 17-3 1.0% Pro and 0.5% FOS-containing PBS solution Example 18-1 0.1% Thr and 0.5% FOS-containing PBS solution Example 18-2 0.5% Thr and 0.5% FOS-containing PBS solution Example 18-3 1.0% Thr and 0.5% FOS-containing PBS solution Comparative 0.5% FOS-containing PBS solution Example 11 Comparative 1.0% Asn-containing PBS solution Example 12 Comparative 1.0% Cys-containing PBS solution Example 13 Comparative 1.0% His-containing PBS solution Example 14 Comparative 1.0% Leu-containing PBS solution Example 15 Comparative 1.0% Met-containing PBS solution Example 16 Comparative 1.0% Phe-containing PBS solution Example 17 Comparative 1.0% Pro-containing PBS solution Example 18 Comparative 1.0% Thr-containing PBS solution Example 19 Reference PBS alone (control) Example 1

Using the results obtained by quantitative PCR, the relative amount when the control is 1 was calculated, and the results were normalized with z-score.

The results are shown in FIG. 1-2. The data shows a mean of z-score±standard error (n=3).

As a result, proliferation of bifidobacteria was not observed when any amino acid was added alone. However, when His and Pro were respectively added together with 0.5% FOS, greater proliferation of bifidobacteria than that with FOS alone was observed.

Thus, a synergistic effect exceeding the additive effect was observed in the proliferation of bifidobacteria by combining an amino acid and an oligosaccharide.

Experimental Example 2: Addition Test of Amino Acid and Galacto-Oligosaccharide to Human Feces Suspension

The PBS solutions for the Examples and Comparative Examples shown in Table 2 were prepared and used as media for a feces culture. Feces culture and detection of bifidobacteria were performed according to methods similar to those in Experimental Example 1-1. Galacto-oligosaccharide (GOS) manufactured by Wako Pure Chemical Industries, Ltd. (#076-05945) was used.

TABLE 2 medium for feces culture Example 19-1 0.1% Glu and 0.5% GOS-containing PBS solution Example 19-2 0.5% Glu and 0.5% GOS-containing PBS solution Example 19-3 1.0% Glu and 0.5% GOS-containing PBS solution Example 20-1 0.1% Lys and 0.5% GOS-containing PBS solution Example 20-2 0.5% Lys and 0.5% GOS-containing PBS solution Example 20-3 1.0% Lys and 0.5% GOS-containing PBS solution Example 21-1 0.1% Glu, 0.1% Lys and 0.5% GOS-containing PBS solution Example 21-2 0.5% Glu, 0.5% Lys and 0.5% GOS-containing PBS solution Example 21-3 1.0% Glu, 1.0% Lys and 0.5% GOS-containing PBS solution Comparative 0.1% Glu-containing PBS solution Example 20-1 Comparative 0.5% Glu-containing PBS solution Example 20-2 Comparative 1.0% Glu-containing PBS solution Example 20-3 Comparative 0.1% Lys-containing PBS solution Example 21-1 Comparative 0.5% Lys-containing PBS solution Example 21-2 Comparative 1.0% Lys-containing PBS solution Example 21-3 Comparative 0.1% Glu and 0.1% Lys-containing Example 22-1 PBS solution Comparative 0.5% Glu and 0.5% Lys-containing Example 22-2 PBS solution Comparative 1.0% Glu and 1.0% Lys-containing Example 22-3 PBS solution Comparative 0.5% GOS-containing PBS solution Example 23 Reference PBS alone (control) Example 2

Using the results obtained by quantitative PCR, the relative amount when the control is 1 was calculated, and the results were normalized with z-score.

The results are shown in FIG. 2. The data shows a mean of z-score±standard error (n=3).

As a result, proliferation of bifidobacteria was not observed when amino acid was added alone at any concentration. However, when Glu and Lys were respectively added together with 0.5% GOS, greater proliferation of bifidobacteria than that with GOS alone was observed. In addition, when Glu and Lys were mixed and added together with 0.5% GOS, greater proliferation of bifidobacteria than that with GOS alone was similarly observed.

Thus, a synergistic effect exceeding the additive effect was observed in the proliferation of bifidobacteria by combining an amino acid and an oligosaccharide irrespective of the type thereof.

Experimental Example 3: Administration Test of Enteric Lys and FOS to Pigs

Six 2 to 3 months-old male pigs (HI-COOP SPF pig LWD) were purchased from ZEN-NOH, divided into 2 test groups each containing 3 pigs such that the average body weight was uniform and kept for 4 weeks at 3 pigs/pen. In each test group, 400 g/head of pig MIRAI CEX (ZEN-NOH) was fed twice per day. In each test group, the test substance was administered by the dosage and method shown in Table 3. A lysine preparation “AjiPro-L” for dairy cows manufactured by Ajinomoto Co., Inc. (lysine hydrochloride content is 40% or more) was used as Enteric Lys, and fructo-oligosaccharide “Mayoligo P granule” manufactured by Meiji Food Materia Co., Ltd. (FOS content is 95% or more) was used as FOS. After 4 weeks from the start of administration, the cecum was collected, and bifidobacteria in the cecal contents were examined by quantitative PCR in the same manner as in Experimental Examples 1-1, 1-2 and 2. The concentration of Lys in the cecum contents was measured using 6470 Triple Quad LC/MS/MS system manufactured by Agilent and Intrada Amino Acid column manufactured by Imtakt.

TABLE 3 dosage and administration method of test substance Example 22 FOS: Meiorigo P granule (10 g) was mixed with each meal Enteric Lys: Oral administration of 2 cellulose capsules after each meal, each capsule encapsulating 6 g of AjiPro-L Comparative FOS: Meiorigo P granule (10 g) was mixed with each meal Example 24 placebo: Oral administration of 2 empty cellulose capsules after each meal

The results obtained by quantitative PCR are shown as relative amounts when Comparative Example 24 (placebo) is 1. The results of the relative amount of bifidobacteria in the cecum are shown in FIG. 3 (mean±standard deviation, n=3). As a result, pig intestinal bifidobacteria proliferation is about 3 times higher on average when the combination of enteric Lys and FOS was ingested than by the ingestion of FOS alone.

In addition, the results of the concentration of Lys in the cecum at that time are shown in FIG. 4 (mean±standard deviation, n=3). As a result, it was confirmed that the concentration of Lys in the cecum increased in the enteric Lys ingestion group.

Thus, a synergistic effect with fructo-oligosaccharide was observed by increasing the concentration of Lys in the lower gastrointestinal tract by the ingestion of enteric Lys. 

1. A feed composition for improving an intestinal environment comprising: (1) an enteric composition comprising an amino acid or a salt thereof, and (2) an indigestible polysaccharide or a composition comprising an indigestible polysaccharide.
 2. The feed composition according to claim 1, wherein the indigestible polysaccharide is an oligosaccharide.
 3. The feed composition according to claim 1, wherein the amino acid is selected from the group consisting of alanine, glutamine, glutamic acid, isoleucine, histidine, lysine, proline, valine, and combinations thereof.
 4. The feed composition according to claim 1, wherein the amino acid is selected from the group consisting of glutamic acid, lysine, and a combination thereof.
 5. The feed composition according to claim 1 wherein the form of the enteric composition is selected from the group consisting of a granule, a granulated substance, a tablet, a hard capsule, and a soft capsule.
 6. A method for improving intestinal environment in a non-human mammal, bird, fish, or crustacean comprising ingestion by the non-human mammal, bird, fish, or crustacean of an effective amount of a feed composition comprising: (1) an enteric composition comprising an amino acid or a salt thereof, and (2) an indigestible polysaccharide or a composition comprising an indigestible polysaccharide.
 7. A method for producing a feed composition for improving the intestinal environment comprising combining (1) an enteric composition comprising an amino acid or a salt thereof, and (2) an indigestible polysaccharide or a composition comprising an indigestible polysaccharide. 